Bacteriorhodopsin is a rhodopsin-like protein. It functions as the light receptor for photosynthesis in halobacteria and is localized in the purple membrane, which forms part of the cell membrane. In the light bacteriorhodopsin generates an electrochemical gradient by transferring protons from the cell interior into the suspending medium. Energy stored in the gradient is used by the cells for ATP synthesis and other energy-requiring processes. We propose to investigate the kinetics of the photoreaction cycle in bacteriorhodopsin in isolated purple membrane, in isolated cell envelopes and in intact cells to grain insight into the proton-pumping mechanism. Mainly flash and low-temperature spectroscopy will be used for this purpose. Proton release and uptake will be followed by adding pH indicator dyes and following their absorption or fluorescence changes. Bacteriorhodopsin preparations modified through detergent or organic solvent treatment or chemically modified will also be used. The energy coupling mechanisms in intact cells will be established by measuring electric membrane potentials, pH and other ion gradients in the light and in the dark. This will be accomplished with the use of radioactive tracers, measuring the distribution of physiologic ions and added permeable ions, weak acids and bases. Kinetics of the changes will be followed with ion specific electrodes and indicator dyes. Photophosphorylation will be studied by monitoring ATP synthesis with a time resolution of 1.0 sec or better together with one or more of the other relevant parameters such as pH gradient or membrane potential changes. Isolated cell envelopes will be studied with the same techniques to measure the same parameters under better-defined conditions. Microcalorimetry at high time resolution will be attempted to establish the thermodynamics of the systems.